PROJECT 1: ESTABLISHING STABLE MIXED HEMATOPOIETIC CHIMERISM INA CANINE MODEL We have established stable dog leukocyte antigen (DLA)-identical marrow grafts using sublethal conditioning with 2 Gy total body irradiation (TBI) before and a short course of immunosuppression with mycophenolate mofetil and cyclosporine after transplantation. The approach has been translated successfully into the clinic to treat patients with malignant (see Projects 3 and 4) and nonmalignant blood disorders. Project 1 will use the canine model to address three major issues of clinical allogeneic hematopoietic cell transplantation (HCT), avoiding long-term sequelae from conditioning regimens, controlling graft-vs.-host disease (GVHD) without need for and side effects from extended postgrafting immunosuppression, and enhancing graft-vs.- tumor effects without increasing the risk of GVHD. As for the first goal, we have both preclinical and clinical evidence that cytotoxic conditioning is not mandatory for allogeneic grafts to home. We will extend these observations and determine whether specific and non-toxic pretransplant tolerance induction, exploring six canine-specific blockers of T-cell co-stimulation and two agents affecting regulatory pathways, developed in our laboratory, can be substituted for the broad immunosuppression imparted by TBI and, this way, avoid short- and long-term radiation toxicities. As for the second goal, we will assess three alternative postgrafting manipulations for better control of both GVHD and HVG reactions which, if successful, would avert the need for extended postgrafting immunosuppression. To that end, we will study the immunosuppressive agent cyclophosphamide administered 3 days after marrow grafting, an astatine-211 (211At)-labeled monoclonal antibody against the T-cell activation antigen CD70, and an antagonist to the T-cell costimulatory blocker CD28 combined with an agonist to the down-regulatory molecule CTLA-4. The studies proposed under the first two goals are likely to generate stable mixed donor/host hematopoietic chimeras. Persistent host hematopoiesis can serve as model of persistent hematologic malignancy after HCT, a frequent problem encountered in patients transplanted under Projects 3 and 4. In collaboration with Project 2, we intend to identify minor non-DLA antigen disparities specific for hematopoietic cells (hematopoietic antigens) in given donor/recipient pairs using genomics approaches. This knowledge will provide the basis for future studies addressing the third goal in which donor lymphocytes rendered immune to host hematopoietic antigens will be infused in order to shift mixed to all-donor chimerism with no or minimal GVHD. If successful, we will test the efficacy of this approach in a canine model of experimentally induced acute leukemia. Results of these preclinical studies will be relevant for future clinical trials under Projects 3 and 4 and are likely to increase success and safety of allogeneic HCT in human patients.